Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
  • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
  • G01N2021/6441—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N2021/6463—Optics
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N2021/6463—Optics
  • G01N2021/6471—Special filters, filter wheel
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
  • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
  • G01N21/64—Fluorescence; Phosphorescence
  • G01N21/645—Specially adapted constructive features of fluorimeters
  • G01N2021/6482—Sample cells, cuvettes

Definitions

• the present invention measures the intensity of transmitted light or emitted light for each dye when a sample in which a plurality of dyes are mixed is irradiated with light having a wavelength corresponding to each dye.
• a sample in which multiple fluorescent dyes are mixed is irradiated with light at the excitation wavelength of each fluorescent dye, and the fluorescence excited by these lights is measured.
• the present invention relates to a fluorescence measurement device and a fluorescence measurement method.
• a sample mixed with a dye is irradiated with light having a wavelength corresponding to the dye.
• the dye-labeled substance is detected by calculating the absorbance by measuring the intensity of the transmitted light.
• a dye-labeled substance is detected by calculating the reflectance by measuring the intensity of scattered light instead of transmitted light.
• a plurality of different types of dyes are mixed with the sample according to the substance to be detected, and light corresponding to each dye is separately applied to the sample. Irradiated.
• a plurality of dyes fluorescent dyes
• a plurality of dyes having different excitation wavelengths and fluorescence wavelengths are mixed.
• Each sample is separately irradiated with light of the excitation wavelength of each dye, and the component analysis is performed by measuring the fluorescence intensity of each dye.
• the absorbance measurement a sample in which a plurality of dyes having different absorption wavelengths are mixed is irradiated with light of the absorption wavelength of each dye separately, and the intensity of the transmitted light is measured for each dye. A component analysis is performed.
• the excitation wavelength, absorption wavelength, and reflection wavelength of the dye usually have a certain width. For this reason, when fluorescence measurement is used, if the excitation peak wavelengths are close to each other between the used dyes (fluorescent dyes), when one dye is excited by light of the excitation wavelength, the other dyes are also excited. In some cases. In this case, the obtained fluorescence intensity becomes a composite value of the fluorescence intensity of each excited dye, and it is difficult to perform accurate component analysis, genetic diagnosis, and the like.
• An object of the present invention is to provide a measuring apparatus and a fluorescence measuring method capable of separating and measuring the actual intensity of each dye from the combined value of the intensity of transmitted light and emitted light. Disclosure of the invention
• a measuring device is a measuring device that measures the intensity of transmitted light or emitted light when irradiating light having different wavelengths to a sample in which a plurality of dyes are mixed, for each of the dyes.
• a light source unit that can irradiate the sample, a light receiving unit that receives the transmitted light or the emitted light, and outputs an electric signal according to the intensity of the received light, and a computing unit. Any one of the plurality of dyes is mixed, and the light receiving unit outputs when the light source unit irradiates light having different wavelengths to each of the plurality of correction samples in which the mixed dyes are different from each other.
• the intensity of the transmitted light or the emitted light is calculated for each of the dyes using a correction coefficient calculated based on the electrical signal to be generated.
• the sample may include a mixture of a plurality of fluorescent dyes having different excitation wavelengths as the dye, wherein the light receiving unit receives the fluorescent light by the fluorescent dye, and receives the fluorescent light.
• the arithmetic unit outputs an electric signal according to the fluorescence intensity, and the arithmetic unit performs the processing for each of the plurality of correction samples in which one of the plurality of fluorescent dyes is mixed and the mixed fluorescent dyes are different from each other.
• the light source unit emits light having the excitation wavelength of each of the plurality of fluorescent dyes
• the fluorescence of the fluorescence emitted from the sample is calculated using a correction coefficient calculated based on an electric signal output by the light receiving unit.
• a mode in which the fluorescence intensity for each dye is calculated may be used.
• the measuring device according to the present invention functions as a fluorescence measuring device.
• the plurality of fluorescent dyes mixed with the sample are numbered 1 to n, and the light source unit is k (1, 2, ..., n).
• the output value of the electric signal output from the light receiving unit when the sample is irradiated with light of the excitation wavelength of the fluorescent dye No. X is X k , and the fluorescence intensity of the fluorescent dye No.
• the fluorescence intensity YiYn is calculated as the fluorescence intensity of each of the fluorescent dyes by substituting the output value XiXn.
• the light source monitor has a light amount monitor that detects a light amount of light emitted from the light source unit and outputs a signal to the calculation unit. Based on the output signal, the output value ⁇ . Or also a preferred embodiment for correcting the matrix elements aii ⁇ a nn.
• a fluorescence measuring method comprises: a light source unit capable of irradiating light having different wavelengths; A fluorescence measurement method for measuring the fluorescence intensity of each of the fluorescent dyes emitted from a sample in which a plurality of fluorescent dyes having different excitation wavelengths are mixed, using a light receiving unit that outputs an electric signal, Calculating a fluorescence intensity of the fluorescence emitted from the sample for each of the fluorescent dyes using a correction coefficient, wherein the correction coefficient is any one of the plurality of fluorescent dyes, and The light receiving unit is output when the light source unit irradiates each of the plurality of correction samples in which the mixed fluorescent dyes are different from each other with light having an excitation wavelength of each of the plurality of fluorescent dyes.
• the plurality of fluorescent dyes mixed with the sample are numbered l to n, and the light source unit is a k (1,2, ..., n) fluorescent dye.
• the values of the fluorescence intensities ⁇ to ⁇ are calculated as the fluorescence intensities of the respective fluorescent dyes.
• the present invention may be a program for realizing the above-described fluorescence measuring method according to the present invention. By installing this program on a computer and executing it, the fluorescence measurement method according to the present invention can be executed.
• “dye” includes fluorescent dyes used in fluorescence measurement in addition to dyes used in absorbance measurement and reflectance measurement. When only a fluorescent dye is meant among “dyes”, it is referred to as “fluorescent dye”.
• FIG. 1 is a configuration diagram showing a fluorescence measurement device which is one embodiment of the measurement device of the present invention.
• FIG. 2 is a flow chart showing a fluorescence measurement process performed by the fluorescence measurement device shown in FIG.
• FIG. 3 is a flowchart showing a correction coefficient calculating process performed by the fluorescence measuring device shown in FIG.
• FIG. 4 is a flowchart showing a light amount correction value calculation process performed by the fluorescence measuring device shown in FIG.
• FIG. 1 is a configuration diagram showing a fluorescence measurement device which is one embodiment of the measurement device of the present invention.
• the fluorescence measurement device shown in FIG. 1 is a device for measuring the fluorescence intensity of each fluorescent dye emitted from the sample 6. As shown in FIG. 1, the fluorescence measuring device includes a light source unit 1, a light receiving unit 2, a calculation unit 3, a display unit 4, a reaction vessel 5, and a light amount monitor 7.
• a sample 6 in which a plurality of fluorescent dyes are mixed is added to the reaction vessel 5.
• the sample contains four types of fluorescent dyes shown in Table 1 below.
• the fluorescent dye mixed with the sample is not limited to those shown in Table 1 below, and the number thereof is not limited.
• a necessary number of appropriate fluorescent dyes can be selected according to the purpose of the fluorescence measurement and the like.
• the light source unit 1 is configured to be capable of irradiating light having different wavelengths, and can irradiate light having an excitation wavelength of a fluorescent dye mixed with a sample.
• the light source unit 1 includes light emitting elements 11 a to 11 d, dichroic mirrors 12 a to 12 d, and a total reflection mirror 13.
• the light emitting elements 11 a to 11 d emit light in accordance with an instruction from the arithmetic unit 3 and emit light for exciting the fluorescent dye mixed in the sample 6.
• the light emitting elements 11a to lId are arranged such that the emission directions of the light emitting elements are parallel.
• the wavelengths of light emitted from the light emitting elements 11a to 11d are different from each other, and are set to one of the excitation wavelengths of the fluorescent dye mixed in the sample.
• the light-emitting element 11a emits light at the FAM excitation wavelength
• the light-emitting element 11b emits light at the J ⁇ E excitation wavelength
• the light-emitting element 11c emits light at the TAMRA excitation wavelength.
• the light emitting element 11 d emits light having an excitation wavelength of ROX.
• the dichroic mirrors 12a to 12d have a characteristic of reflecting only light having a wavelength equal to or less than a specific wavelength (high-pass), and the dichroic mirrors 12a, 12b, 12c, and The maximum wavelength of light that can be reflected increases in the order of 1 2 d.
• the light emitted from each of the light-emitting elements 11 a to 11 d enters the total reflection mirror 13 through the same optical path, is reflected by this, and enters the reaction vessel 5.
• the light amount of the light emitted from the light emitting elements 11 a to 11 d is monitored by the light amount monitor 7.
• the light amount monitor 7 detects the light amount of the light emitted from the light emitting elements 11 a to 11 d and outputs a signal to the calculation unit 3. Note that, in the example of FIG. 1, since the four types of fluorescent dyes are used as described above, the number of light emitting elements constituting the light source unit 1 is also four. Also, the number of dichroic mirrors is four according to the number of light emitting elements.
• the number of light emitting elements and dichroic mirrors is not limited to this, and is determined according to the number of fluorescent dyes used.
• the luminous element It is preferable to use a light-emitting diode semiconductor laser as the elements 11a to 11d, but it is also possible to use a xenon lamp or a halogen lamp.
• the light receiving unit 2 receives the fluorescence emitted from the reaction container 5 and outputs an electric signal corresponding to the fluorescence intensity of the received fluorescence.
• the light receiving unit includes light receiving elements 14 a to 14 d, dichroic mirrors 15 a to 15 d, and a total reflection mirror 16.
• the dichroic mirrors 15a to 15d have a characteristic of reflecting (low-pass) only light having a wavelength longer than a specific wavelength.
• the minimum wavelength of the light that can be reflected increases in the order of 15c, 15b, and 15a.
• the light receiving elements 14a to 14d are photo diodes, and are arranged such that light reflected by one dichroic mirror is incident on a light receiving surface (not shown) of one light receiving element.
• the fluorescence emitted from the reaction vessel 5 is reflected by the total reflection mirror 16 and then reflected by one of the dichroic mirrors 15a to 15d according to the wavelength, and the corresponding light is received. It will be incident on the element. As a result, an electric signal corresponding to the fluorescence intensity of the fluorescence is output from each light receiving element to the arithmetic unit 3.
• the calculation unit 3 calculates the fluorescence intensity based on the electric signal output from the light receiving unit 2. The calculated result is displayed on the display unit 4.
• the display unit 4 is a liquid crystal display, a CRT, or the like.
• the fluorescence measurement method of the present invention will be described with reference to FIGS. Note that the fluorescence measurement method of the present invention can be executed by operating the fluorescence measurement device shown in FIG. Therefore, in the following description, the operation of the fluorescence measurement device shown in FIG. 1 will be described.
• FIG. 2 is a flowchart showing a fluorescence measurement process performed by the fluorescence measurement device shown in FIG. As shown in Fig. 2, first, the calculation of the fluorescence measurement device The unit 3 determines whether a correction coefficient has been calculated (step S 1). The correction coefficient is used to calculate the fluorescence intensity of each fluorescent dye from the electrical signal output by the light receiving unit 2 when irradiating the sample containing multiple fluorescent dyes with light of the excitation wavelength of each fluorescent dye. It is a coefficient.
• the term “fluorescence intensity for each fluorescent dye” does not refer to a composite value obtained by conventional fluorescence measurement, but the wavelength of the light and the excitation wavelength when the sample is irradiated with light. It refers to the fluorescence intensity of only the fluorescence emitted by the corresponding fluorescent dye. That is, in the present invention, the actual fluorescence intensity is separated from the composite value by using the correction coefficient as described later.
• the correction coefficient is a row example (a.) Satisfying the above equation (1).
• the four types of fluorescent dyes mixed with the sample are numbered from 1 to 4 in ascending order of excitation wavelength, and light source unit 1 excites the fluorescent dye of k (1, 2, 3, 4).
• step S2 If it is determined in step S1 that the correction coefficient has not been calculated yet, the calculation unit 3 performs a correction coefficient calculation process (step S2), and then executes the following step S3. The specific contents of the correction coefficient calculation processing in step S2 will be described later.
• the arithmetic unit 3 emits light from each of the light emitting elements 11 a to l 1 d and measures the light amount by the light amount monitor 7, If there is a change in the measured amount of light, it calculates the light quantity correction value for correcting the output value Xi ⁇ X 4 described later (step S 3). The specific contents of step S3 will be described later.
• the arithmetic unit 3 causes each light emitting element of the light source unit 1 to irradiate the sample with light having an excitation wavelength to measure the fluorescence intensity (step S4).
• the output value of the electric signal output from the light receiving unit 2 is obtained by performing I / V conversion on the current value of the electric signal output by the light receiving elements 14a to 14d.
• the output value of the electric signal output from the light receiving unit 2 may be a digital value obtained by A / D converting the current value of the electric signal output from the light receiving elements 14a to 14d.
• Step S 6 the operation unit 3 executes the steps S 4 and S 5 again.
• step S7 the calculation unit 3 substitutes the correction coefficient and the output value obtained in step S5 into the above equation (2) to obtain each fluorescent dye.
• Step S7 the calculation unit 3 substitutes the correction coefficient and the output value obtained in step S5 into the above equation (2) to obtain each fluorescent dye.
• Step S7 Calculate the fluorescence intensity Yi Y (step S7).
• the fluorescence measurement process is completed, and the fluorescence intensity of each fluorescent dye is displayed on the display.
• the actual fluorescence intensity can be separated from the synthesized value, so that more accurate fluorescence measurement can be performed as compared with the related art.
• FIG. 3 is a flowchart showing a correction coefficient calculation process performed by the fluorescence measurement device shown in FIG.
• the correction coefficient calculation process is performed using a plurality of correction samples.
• the correction sample is a mixture of only one of the fluorescent dyes mixed with the sample, and each correction sample is mixed with a different fluorescent dye. That is, in the example of FIG. 3, as shown in Table 1, four types of fluorescent dyes are mixed with the sample, so that four types of correction samples are required.
• the correction coefficient calculation process is performed using the following equations (3) to (6) obtained by expanding equation (2).
• the calculation unit 3 irradiates the correction sample with light from each light emitting element (step S11), and further outputs the electric signal output by the light receiving unit 2 at this time. Obtain the value (step S1 2).
• the arithmetic unit 3 substitutes the obtained output value into the above equations (3) to (6) (step S13).
• step S14 determines whether output values have been obtained for all the correction samples. If output values have not been obtained for all correction samples, steps S11 to S13 are executed again. If output values have been obtained for all correction samples, step S15 is executed.
• Steps S11 to S13 will be specifically described.
• the calculation unit 3 irradiates the correction sample in which only the first fluorescent dye (FAM) is mixed with light having the excitation wavelength of the first to fourth fluorescent dyes by the light source unit 1.
• the arithmetic unit 3 calculates the output values F1 to f in equations (3) to (6) above. Substitute ⁇ F4.
• the calculation unit 3 includes a correction sample in which only the second fluorescent dye (JOE) is mixed, a correction sample in which only the third fluorescent dye (TAMRA) is mixed, and a fourth fluorescent dye.
• the light source unit 1 irradiates light of the excitation wavelength of the No. 1 to No. 4 fluorescent dyes and obtains the output value of the output electric signal, and obtains it.
• the output values obtained are substituted into equations (3) to (6). If the output values in these cases are J1 to J4, T1 to T4, and R1 to R4, respectively, the following equations (11) to (22) are obtained.
• the correction coefficient calculation process ends.
• the correction coefficient calculation process can be performed before the shipment of the fluorescence measurement device.
• it is preferable that the correction coefficient is stored in advance in the memory of the fluorescence measuring device at the stage of shipping the product.
• steps S1 and S2 need not be performed in the fluorescence measurement processing shown in FIG.
• FIG. 4 is a flowchart showing a light amount correction value calculation process performed by the fluorescence measuring device shown in FIG.
• the light intensity of the light emitting element 1 1 a ⁇ l I d is, when variation by change over time or environmental changes,: a 2 1: a 3 1: the ratio of a 41, a i 2 : a 2 2: a 3 2 : ratio of a 42, a i 3: a 2 3: a 3 3: the ratio of a 43, and a 14: a 24: a 34 : a ratio of a 44 is affected, This is performed in order to prevent an inaccurate fluorescent luminance from being calculated.
• the calculation unit 3 first sets the light amount ratio of the light emitting elements 11a, lib, 11c, and 11d when the correction coefficient is determined to 1: 1. : 1: 1 (reference value), the current light intensity ratio of the light emitting element is measured based on the signal from the light intensity monitor (step S21).
• the arithmetic unit 3 determines whether or not the calculated light amount ratio fluctuates from 1: 1: 1: 1 (step S22). If there is no change, the arithmetic unit 3 ends the processing. On the other hand, if it fluctuates, a light amount correction value corresponding to the fluctuation width is calculated (step S23).
• the light amount ratio of the light emitting elements 11a, lib, 11c, and 11d is 1: 2: 3: 4.
• the light intensity of each light emitting element must be (1/1), (1Z2), (1/3) and (1/4) times, respectively. Therefore, the light intensity correction values are (1 1), (1/2), (1/3), (1/4).
• the light amount correction value is preferably determined in consideration of the sensitivity of the light receiving elements 14a to 14d.
• step S7 of FIG. 2 instead of X x ⁇ 2 , ⁇ 3 and ⁇ 4 in the above equation (2), the operation unit 3 replaces (1/1) X or (1/2) ⁇ 2 , Substitute (1/3) X 3 and (1 Z4) X 4 to calculate the fluorescence intensity ⁇ to ⁇ .
• the fluorescence intensity can be calculated by correcting the light intensity of the light emitting element.
• the accuracy of the measurement can be further improved.
• a program for implementing steps S1 to S7 shown in FIG. 2 is installed in a computer connected to the light source unit 1 and the light receiving unit 2, and the program is executed.
• the CPU central processing unit
• the CPU central processing unit of the computer functions as the arithmetic unit 3.
• the fluorescence measurement device and the fluorescence measurement method are described as examples.
• the present invention is not limited to this example, and may be a measuring device or a measuring method using absorbance measurement or reflectance measurement. That is, according to the present invention, even when a plurality of dyes having different absorption wavelengths or reflection wavelengths are mixed in a sample, the correction coefficient can be calculated in the same manner as described above, and transmitted light or scattered light can be calculated. Can be calculated for each dye. Also, the absorbance can be calculated from the calculated transmitted light intensity, and the reflectance can be calculated from the scattered light intensity.
• the measurement apparatus and the fluorescence measurement method of the present invention the Even when a number of dyes, for example, fluorescent dyes, are mixed, the actual intensity of each dye can be separated from the obtained intensity of transmitted light or emitted light (fluorescence). Therefore, by using the measuring apparatus and the fluorescence measuring method of the present invention, it is possible to perform more accurate component analysis, genetic diagnosis, and the like than before.
• a number of dyes for example, fluorescent dyes

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